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. 2008 Nov 4;17(1):121–130. doi: 10.1038/mt.2008.224

Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells

Trine Dalsgaard 1, Brian Moldt 1, Nynne Sharma 1, Gernot Wolf 1, Alexander Schmitz 2, Finn S Pedersen 2, Jacob G Mikkelsen 1
PMCID: PMC2834987  PMID: 18985029

Abstract

The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector–containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5′-HS4 chicken β-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.

Introduction

With the revived Sleeping Beauty (SB) DNA transposon as a driving force,1 transposable DNA elements have emerged as new promising integrating gene vehicles with applications ranging from transgenesis2 and insertional mutagenesis3,4,5 to therapeutic gene transfer.6 The simple gene integration machinery of cut-and-paste DNA transposons is based on the specific binding of transposase subunits to inverted repeat sequences flanking one or more gene cassettes of interest. Nonviral vector systems have traditionally relied on random insertion or suffered from short-term gene expression from episomal plasmid DNA. By taking advantage of transposase-directed gene insertion, nonviral carriers become actively and efficiently inserted in the genome. The potential for achieving long-term transgene expression with such vectors has been demonstrated in liver,6,7,8,9,10 lung,11,12 and brain13,14 of adult mice injected with plasmid DNA.

DNA transposable elements appear to have colonized almost every living organism. Elements that were once active and able to colonize and spread in new hosts have accumulated mutations during evolution and the far majority of these elements now exist as fossil remnants of the past. To combat potential negative effects of actively mobilizing DNA transposons, mechanisms for suppression have evolved. These include inhibition of transposition by transposase overproduction,15,16 RNA interference (RNAi),17 and epigenetic regulation including DNA methylation and histone modifications.18 These mechanisms of regulation may apply also to DNA transposon–based vectors and their use in vivo. Hence, we have previously shown that transposition of SB vectors in mouse liver is inhibited by high levels of transposase8 and have found indications that gene-regulatory activities in the terminal regions of SB can direct production of complementary RNAs with the potential of inducing an RNAi response against the transposon.19,20 Moreover, we have provided evidence that SB vectors may be subjected to transcriptional silencing in human cells.21

In this article, we examine transcriptional silencing of SB-based vectors in murine F9 embryonal carcinoma (F9 EC) cells. F9 EC cells are recognized as a relevant model system to study growth and differentiation during mammalian embryogenesis. On treatment with retinoic acid the cells differentiate into primitive endoderm22 and by further treatment with cyclic AMP into parietal endoderm.23 Previous work has demonstrated that replication of Moloney murine leukemia virus in F9 EC cells is restricted because of potent silencing of transcription from the viral promoter.24,25 Repression is caused by recruitment of the transcriptional corepressor TRIM28 to the proline primer-binding site (PBS) of Moloney murine leukemia virus leading to dimethylation of histone H3 lysine 9 and recruitment of HP1γ.26,27 Interestingly, the repressive state can be relieved by introducing mutations in the PBS sequence24 or by substituting the PBS with other natural or synthetic PBS sequences.28 In addition to the immediate and potent TRIM28-directed response, DNA methylation appears to play a role in further silencing during differentiation of the EC cells.29 Adding to these silencing pathways, double-stranded RNA induces a strong RNAi response in F9 cells leading to robust knockdown of both endogenous and exogenous genes.30 In a study performed exclusively in human HeLa cells, integrated SB transposons carrying a transgene driven by the promoter of Rous sarcoma virus (RSV) were frequently silenced. Hence, 13 of 17 clones containing a single transposon insertion were completely silenced during the course of the experiment.21 Although such silencing may be influenced by the chromatin context at the insertion site, it was suggested that CpG methylation also played a key role in postintegrative gene silencing of these vectors. In this article, we tested the performance of such vectors in F9 EC cells and use our findings as a model system for creation of SB-derived vectors that are protected against transcriptional silencing.

Insulators are DNA elements that can serve as genetic barriers to chromosomal position effects allowing protection against spreading of condensed inactive chromatin. Some elements may in addition serve to block the molecular communication between enhancers and genes. The 5′-HS4 chicken β-globin insulator (cHS4) is located at the 5′ boundary between heavily condensed chromatin and the actively transcribed β-globin gene in chicken erythroid cells31 and has been found to possess both barrier- and enhancer-blocking effects. The 1.2-kb cHS4 element has been widely used in the context of retroviral vectors leading to an increased probability that randomly inserted vectors will express, as demonstrated by increased stability of gene expression in murine erythroleukemia cells32 and in mice transplanted with transduced bone marrow.33,34 The barrier function of cHS4 is coupled to H3 hyperacetylation and reduced H3 methylation of histones adjacent to the insulator as well as to a concomitant reduction in CpG methylation.35,36,37,38,39,40 This scenario is presumed to form the basis for the ability of cHS4 to protect against silencing of nearby promoters38 also within the context of retroviral vectors.35 The specialized chromatin structure (scs) from Drosophila has been found to block repression mediated by chromatin-associated repressors in human U2-OS cells41 but insulator activity for scs could not be detected in a colony assay in human K562 cells,31 and only the full-length scs/scs′ motif is functional after vertical transmission in zebrafish.42 The scs sequence functions in enhancer blocking in Xenopus,43 as well as in human Jurkat cells.44 However, attempts to shield internal promoters in retroviral vectors against PBS-mediated transcriptional repression with cHS4 and scs have been unsuccessful.45

Promoter silencing and the protective functions of insulators vary between cell types. Using both a nonselective fluorescence-activated cell sorter–based method and an antibiotic selection scheme, we investigate here whether SB vectors, unlike gammaretroviral vectors, are able to escape silencing in a model system based on F9 EC cells. In a search for potential protective cis-elements in the terminal regions of SB, we show, nevertheless, that transgene cassettes in the context of integrated SB DNA transposon–based vectors are in fact efficaciously silenced in early embryonal cells. Our findings demonstrate that SB vectors containing 1.2-kb cHS4 insulators flanking the transgene cassette are protected against transcriptional silencing and that transposition of insulated transposons relative to uninsulated transposons results in a significantly increased stable transfection rate in F9 cells.

Results

Robust SB transposition in F9 EC cells

Using a standard transposition assay based on antibiotic selection, we initially examined the SB transposition rate in F9 EC cells and compared it with the ability of SB to confer stable transfection in HeLa cells. Plasmid DNA containing the puromycin resistance gene (puro) driven by a phosphoglycerate kinase (PGK) promoter within the context of a SB transposon (pSBT/PGK-puro) was co-transfected with helper plasmid encoding either the HSB3 transposase or a mutated, inactive variant (mSB), and puromycin-resistant colonies were counted. As shown in Figure 1, the number of resistant colonies obtained with the active transposase in F9 cells was reduced eightfold relative to the amount of colonies obtained in HeLa cells. However, the presence of the active transposase increased the stable transfection rate tenfold over background in F9 cells, indicating that the vector transposed robustly in these cells. The transposition rate is routinely very high in HeLa cells compared to mouse cell lines46 and a certain difference in efficacy was therefore expected. Moreover, the low transfection rates in F9 cells, resulting in transfection of ~1–3% of the cells, contributed to a lower stable transfection rate in F9 cells and a marked difference between the two cell lines. Nevertheless, in this and other experiments we consistently observed that the random insertion rate obtained in the presence of mSB was higher in F9 cells than in HeLa cells.

Figure 1.

Figure 1

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Sleeping Beauty transposition leading to stable transfection in HeLa and F9 cells. HeLa and F9 cells were transfected with 1.5 µg pSBT/PGK-puro and 1.5 µg of either pCMV-HSB3 or pCMV-mSB. Stable transfection rates were determined by selecting transfected cells with puromycin for 10 days.

Postintegrative silencing of SB vector–containing clones generated by a nonselective approach

In consideration of the low transfection rate in F9 cells, we decided to use a helper-independent transposon–transposase vector plasmid for optimized transposition rates in transfected cells. This plasmid cis-vector, pSBT/RSV-eYFP.CMV-SB, contains both a SB10 transposase expression cassette driven by the cytomegalovirus (CMV) promoter and a SB transposon containing the eYFP reporter gene driven by an RSV promoter (Figure 2). To generate clonal cell lines without the use of selection, F9 cells were transfected with pSBT/RSV-eYFP.CMV-SB and subsequently flow-sorted based on the transient expression of eYFP from episomal plasmid DNA. Cells with expression above background were seeded at a very low density (<1 cell per well on average) in 96-well dishes (Figure 2) and allowed to expand resulting in a total of 243 individual clones. Each clone was then transferred to six-well dishes and finally passaged in 10-cm dishes. Approximately 2 weeks after the initial transfection (corresponding to at least 20 cell divisions), genomic DNA from each clone was screened by PCR specifically amplifying part of the eYFP gene. Fifty-seven of the 243 clones were found to contain an integrated transposon by this method indicative of an integration efficiency of >23% in transfected F9 cells under the existing conditions.

Figure 2.

Figure 2

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Schematic representation of the experimental setup allowing analysis of transgene expression by a nonselective method. F9 cells were transfected with the pSBT/RSV-eYFP.CMV-SB vector. Forty-eight hours later, the cells were flow-sorted and individually seeded in 96-well dishes (<1 cell per well) based on transient eYFP expression and allowed to grow until colonies developed. Single colonies were transferred to 6-well dishes and further expanded to cell lines. Individual clones were PCR-screened for transposon integration and analyzed by flow cytometry to determine the initial YFP expression level and allow categorization of each clone based on the expression status. Clones were characterized according to which of the following categories the majority of cells were located in: N, negative for expression (100–101); L, low expression (101–102); I, intermediate expression (102–103); H, high expression (103–104).

The expression profile of 27 SB transposon-positive cell lines was determined by flow-cytometric analysis carried out before further passage of the cell lines. A range of different expression profiles, ranging from the absence of detectable eYFP to very strong eYFP expression, were identified (Supplementary Figure S1). Representative examples are shown in Figure 3a. Each cell line was categorized in one of four expression categories (‘N' for negative and ‘L', ‘I', and ‘H' for low, intermediate, and high expressors, respectively; Figure 2) according to the initial measure of eYFP expression. Five of the 27 transposon-positive clones did not express eYFP at this early stage after transfection, whereas five additional clones only contained a minority of cells expressing eYFP and therefore by definition belonged to the N category (Supplementary Figure S1). Each of the 27 cell lines was analyzed by Southern blotting (Figure 3b) to determine the number of SB vector integrations per cell line. Despite the low transfection rate only 8 of the 27 cell lines were found to contain a single integration (Figure 3c). Twelve cell lines contained two or three integrations, whereas the remaining seven clones contained more than three integrations. Episomal remnants of transfected plasmid DNA, which would show as an expectedly faint 6-kb band, were not in general detected by the Southern blot analysis. However, a distinct band at this position was detectable in one clone (clone 389) and, thus, it cannot be formally excluded that this clone contained episomal input DNA or an integrated concatamer. Of the eight clones containing one insertion, five did not express eYFP, whereas the remaining three clones had low expression of the transgene (Figure 3c). In addition, two clones with two insertions and two clones with three insertions were negative for eYFP expression, whereas one clone with multiple insertions (>6) surprisingly turned out to be negative for eYFP.

Figure 3.

Figure 3

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Silencing of the eYFP transgene carried by Sleeping Beauty (SB) transposon-based vectors in F9 cells. (a) Initial eYFP expression profiles in representative F9 clones. Clone 34 and 222 were found to have an intermediate expression level; clone 97, 238, and 389 showed no expression, whereas the majority of YFP positive cells of clone 145 were located in the low-expression category. N, negative for expression (100–101); L, low expression (101–102); I, intermediate expression (102–103); H, high expression (103–104). (b) Determination of copy number of inserted SB vectors by Southern blot analysis. Genomic DNA of transposon-positive clones was digested with EcoRI (indicated by arrows, left panel), which cuts on both sides of the left inverted repeat and within the RSV promoter (indicated by ‘E'). Hybridization was carried out using a probe specific for the eYFP gene. Representative band lengths are indicated by arrows; the mobility of linearized input plasmid (6-kb fragment) is indicated by an asterisk (*). Clone 97 contained one SB insertion, clones 34, 222, and 238 contained two insertions, and clone 389 three vector insertions. (c) Lack of eYFP expression is predominantly observed in clones with low copy number of inserted SB vectors. The initial expression profile of each clone is indicated by the color code for groups of clones containing from one to more than six SB vector insertions. (d) Reignition of eYFP expression by drug treatment. Clones 97, 389, and 478 were treated with a cocktail of 5-azacytidine (5-aza) and TSA to reignite the YFP expression and thereby verify the transposon integration. Clones 97 and 389 were negative for expression immediately after the initial expansion (left panels) and were activated upon drug treatment (right panel). By 5-aza/TSA treatment, eYFP expression was further induced in the positive control clone 478. Initially categorized as an ‘I' clone, clone 478 moved after treatment to the ‘H' category. (e) Expression profiles of six representative clones with initial expression of eYFP. Representative examples illustrate the progression in expression profile over 12 weeks of cell growth. Clone 151, 382, and 490 were silenced over the 12-week period, clone 28 moved slower toward silencing, whereas clones 145 and 478 did not alter expression profile for the duration of the experiment. (f) Schematic representation of the progression of eYFP expression in ten individual clones for the duration of the experiment. Each clone is represented by a dot in the category in which it was categorized according to the initial level of expression. Arrows indicate the mobility of each clone from week 1 to 12. Clones in which expression was stable throughout the experiment are represented by a dot.

To confirm that the eYFP-negative clones indeed contained a functional eYFP-tagged SB transposon vector, all 10 cell clones were treated with 5-azacytidine (5-aza) and trichostatin A (TSA). As shown for three representative clones in Figure 3d, expression of eYFP was efficiently reignited by this treatment in all 10 clones, providing support to the notion that these clones contained one or more functional eYFP expression cassettes that had been silenced during or rapidly after integration. These data suggest that a minimum of 37% of the SB insertions in F9 cells (10 of 27 clones) were subjected to postintegrative silencing shortly after integration. Because the majority of the clones contained more than one insertion, the actual percentage of silenced eYFP insertions could be underestimated by this measure and may more likely be >60%, as suggested by the finding that five of eight single-copy clones were silenced.

Progression of eYFP-positive clones toward silencing

Ten eYFP-positive clones harboring from one to three SB vector integrations were passaged every third day for 12 weeks to investigate their stability of expression over time. Five of these clones were initially categorized as low expressors, whereas the remaining five clones showed intermediate expression. Based on flow-cytometric analysis of cells that had been passaged for 1, 5, 7, and 12 weeks (histogram plot overlays for six representative clones are shown in Figure 3e), five clones (four and one clones from ‘L' and ‘I' categories, respectively) were found to move toward complete silencing, whereas one clone moved from intermediate to low expression. Four clones, all of them containing more than one insertion, maintained stable eYFP expression throughout the experiment (Figure 3f). Three clones that were initially categorized as silenced were passaged in parallel for 7 weeks and showed no evidence of eYFP reignition after prolonged growth (data not shown). In summary, our data demonstrate that the RSV-eYFP cassette, genomically inserted in the context of a SB transposon vector, is frequently silenced in early embryonal cells and that a large proportion of the integrated transposons become transcriptionally silent shortly after insertion.

Increased stable transfection of insulated transposon vectors in F9 cells

To study means of protecting the transposon vector and its transgene cassette from silencing, we set out to test possible protective effects of the cHS4 and a minimal-sized version of the scs derived from Drosophila. In a previous study neither of these insulators was found to protect against PBS-directed silencing of retroviral vectors in F9 cells.45 We first investigated the effect of the insulators in a standard SB transposition assay in which plasmid DNA encoding the SB10 transposase (or the inactive mSB transposase) was co-transfected into F9 cells with plasmid DNA carrying an SB transposon containing a PGK-driven puro gene. On the basis of the standard plasmid pSBT/PGK-puro, we generated pSBT/scs.PGK-puro.scs and pSBT/cHS4.PGK-puro.cHS4 by inserting one copy of the 0.68-kb scs insulator, or the 1.2-kb cHS4 insulator, on each side of the PGK-puro cassette (Figure 4a). To control for the expected negative effect of increased transposon length on transposition, we also constructed pSBT/β-gal.PGK-puro.β-gal in which the PGK-puro cassette in the transposon was flanked by two 1.2-kb sequence fragments derived from internal regions of the β-galactosidase (β-gal) gene.

Figure 4.

Figure 4

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Transposition of insulated SB transposon vectors in F9 cells. (a) Schematic representation of SB transposon vectors. Expression of the puromycin resistance gene (puro) was in all constructs driven by the PGK promoter. The standard gene expression cassette (PGK-puro-pA) was flanked on each side by the 0.6-kb Drosophila scs element (scs), 1.2-kb β-gal sequences (β-gal), the 1.2-kb cHS4 insulator (cHS4), or the 250-bp cHS4 core sequence (cHS4core), as indicated by gray boxes flanking the transgene cassette. Black arrows (labeled IR) indicate the SB inverted repeats. (b) Stable transfection rate of uninsulated and insulated transposons in F9 cells. Transposon vectors (indicated below the x-axis) were co-transfected with plasmid encoding the SB transposase or the inactive mSB variant (gray and white columns, respectively). The stable transfection rate was determined by selecting transfected cells with puromycin and counting the number of resistant colonies. (c) cHS4 core sequences do not support an improved transposon stable transfection rate. Comparison of the effects of the full-length 1.2-kb cHS4 insulator with effects of the short 250-bp cHS4 core sequence. The experiment was performed as described under b. All experiments were performed in triplicates. The significance of differences between relevant groups is indicated by the P values listed above the brackets. (d) Comparable levels of excision of uninsulated and insulated transposon vectors. Transposon excision was measured by a PCR-based excision assay to measure the combined outcome of transposon excision and double-strand DNA repair in the donor plasmid. HeLa cells were transfected with 1 µg SB vector plasmid and 1 µg pCMV-SB10 (or pCMV-mSB, right lane). Consecutive PCRs using sets of primers flanking the transposons were performed to amplify the repaired excision site (lower panel). Plasmid DNA purified from transfected cells was utilized in the first PCR. The identity of the amplification products (~300-bp) was verified by sequence analysis, demonstrating that several different footprints could be identified for each vector. A PCR specific for the Amp gene in the plasmid backbone was utilized to control for the DNA input plasmid (upper panel).

As shown in Figure 4b, transposition from pSBT/PGK-puro resulted in almost 300 puromycin-resistant colonies, whereas about four times fewer colonies were obtained with mSB. For pSBT/scs.PGK-puro.scs and pSBT/β-gal.PGK-puro.β-gal the stable transfection rate was reduced to ~50 and 33%, respectively, relative to the standard transposon vector (P = 0.054 and P = 0.003, respectively). However, when pSBT/cHS4.PGK-puro.cHS4 was transfected along with SB10-encoding plasmid into F9 cells, we registered a significant fivefold increase in the number of clones derived from stable transfection relative to the SBT/PGK-puro standard vector (P = 0.0006) (Figure 4b). Notably, transfection of pSBT/cHS4.PGK-puro.cHS4 produced 15 times more drug-resistant colonies than the pSBT/β-gal.PGK-puro.β-gal control vector harboring a transposon of the exact same length as SBT/cHS4.PGK-puro.cHS4. Furthermore, usage of the cHS4-flanked vector in the presence of mSB resulted in as many colonies as obtained with pSBT/PGK-puro in the presence of SB10 (P = 0.256), indicating that expression also from randomly inserted plasmid vectors was supported by the flanking insulators.

Shorter versions of the cHS4 insulator have previously been shown to direct the protective effects of the element.47 To produce a shorter insulated transposon vector that was expected to be more efficiently transposed, we first replaced each of the 1.2-kb cHS4 insulators flanking the transgene with a single 250-bp cHS4 core sequence which is known to possess enhancer blocking activity but not all the cis-elements for barrier activity.40 As shown in Figure 4c, the resulting vector, pSBT/cHS4core.PGK-puro.cHS4core, did not result in an increased level of stable transfection as compared to the control vector (P = 0.464), and the beneficial effects of the insulators on stable transfection had clearly vanished (P = 0.004, comparison of pSBT/cHS4core.PGK-puro.cHS4core with pSBT/cHS4.PGK-puro.cHS4). We also created a transposon containing the transgene cassette flanked by a 400-bp cHS4 fragment which has previously been shown to facilitate transgene protection in retroviral vectors.47 Again, we did not detect any beneficial effect of these shorter cHS4 elements on the level of stable transfection in F9 cells (data not shown). We conclude from our experiments that 1.2-kb cHS4 insulators flanking the transgene cassette, and not scs insulators or shorter versions of the cHS4 insulator, strongly support stable transfection of F9 cells mediated either by SB transposase-directed gene insertion or randomly inserted plasmid DNA. These findings therefore suggest that the cHS4 insulators may have a beneficial effect on plasmid transfection, transposon mobilization from plasmid DNA, and/or the likelihood of expression from the inserted transgene cassette.

To exclude the formal possibility that the presence of cHS4 insulators in the SBT/cHS4.PGK-puro.cHS4 transposon triggered increased mobilization from transfected donor plasmid, we performed a PCR-based excision assay to measure the combined outcome of transposon excision and double-strand DNA repair in the donor plasmid. Sets of primers flanking the excision site were utilized in a semi-quantitative PCR approach in the linear range to measure footprint product relative to the plasmid DNA input (Figure 4d). Because of the context of the pUC19 plasmid backbone in which the transposon excision site is flanked by LacZ sequences, excision from the negative control vector, pSBT/β-gal.PGK-puro.β-gal, could not be determined by this PCR strategy and was not included in these experiments. However, comparable levels of excision products were detected for each of the vectors pSBT/PGK-puro, pSBT/scs.PGK-puro.scs, and pSBT/cHS4.PGK-puro.cHS4, indicating that transposition was only to a limited degree, if at all, affected by the presence of scs and cHS4 elements. We found indication also of a slightly reduced mobilization of the SBT/cHS4core.PGK-puro.cHS4core transposon, which could have affected the reduced stable transfection rate observed for this particular vector (Figure 4c). In summary, our data suggest that cHS4 insulators in the SB vector context improve the stable transfection rate by increasing the likelihood of expression of the genomically integrated transgene.

Protection of SB vectors from silencing by transgene-flanking cHS4 insulators

The increased colony formation by cHS4-insulated SB vectors prompted us to speculate that these vectors possess a defense against immediate postintegrative silencing. To study the gene expression stability of such SB vectors in more detail, we constructed three SB-based vectors that allowed us to select for successful integration events and subsequently follow the expression profile over time. In these transposition vectors, an RSV-driven bicistronic gene cassette (designated GIP) containing the eGFP reporter gene, an internal ribosomal entry site (IRES), and the puro gene was inserted into the context of SB (Figure 5a). We generated vectors in which the expression cassette was flanked on the left side only by the cHS4 sequence (pSBT/LcHS4.RGIP) or, alternatively, on both sides (pSBT/cHS4.RGIP.cHS4). As shown in Figure 5b, the insulated vector also in this case induced the highest level of stable transfection of F9 cells resulting in a colony count (270 ± 50 colonies) that was more than five times higher (P = 0.004) than in cells co-transfected with pCMV-SB10 and the uninsulated vector, pSBT/RGIP. Moreover, the presence of only one insulator did not prove to have any beneficial effect on the transgene cassette or its mobility as demonstrated by the equal stable transfection rates obtained with pSBT/RGIP and pSBT/LcHS4.RGIP (Figure 5b).

Figure 5.

Figure 5

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Transposition of biscistronic SB vectors in F9 cells. (a) Schematic representation of the transposon vectors, pSBT/RGIP and its derivatives, containing the bicistronic eGFP-IRES-puro (GIP) expression cassette. Expression was driven by the RSV promoter as indicated by the gray arrow in each of the three constructs. The transgene cassette was flanked by the 1.2-kb cHS4 either on the left side of the transgene cassette only (L-cHS4) or on both sides (cHS4). SB inverted repeats (IR) are indicated by black arrows. (b) Stable transfection rate in F9 cells. Transposon vectors (indicated below the x-axis) were co-transfected with plasmid encoding the SB transposase or the inactive mSB variant (gray and white columns, respectively). The stable transfection rate was determined the number of puromycin-resistant colonies appearing after selection. The transfections were performed in triplicates. The significance of differences between relevant groups is indicated by the P values listed above the brackets.

We subsequently set out to follow the expression pattern in transposon-containing clones that had initially been generated by selection with puromycin. Groups of 19, 20, and 24 colonies containing SBT/RGIP, SBT/LcHS4.RGIP, and SBT/cHS4.RGIP.cHS4, respectively, were isolated, expanded, and passaged for 1 week in the presence of puromycin, and then passaged for up to 7 weeks in the absence of puromycin. Each clone was analyzed four times by flow-cytometry during this period resulting in the expression profile overlays shown in Supplementary Figure S2. As an expected outcome of the initial selection for expression of the puro gene, we did not in this setup experience larger variations in reporter gene expression measured immediately after colony expansion. Hence, the majority of all isolated clones (50 of 63 clones) were found to categorize within the group of intermediate expressors at week 1 (Supplementary Figure S2).

Our analysis demonstrated that flanking insulators had a crucial impact on the stability of expression. Hence, approximately half of the clones containing SBT/RGIP and SBT/LcHS4.RGIP were silenced after 7 weeks, whereas only 3 of 24 clones containing SBT/cHS4.RGIP.cHS4 had lost expression (Figure 6a). On the basis of the expression profiles determined at week 1 and 7, each clone was given a score according to its progression from one expression category to another (Figure 6b). Clones with the score −2 had moved two categories toward silencing (from ‘I' to ‘N' or ‘H' to ‘L'), whereas the scores −1 and +1 were given to clones in which reduction or increase of expression corresponded to a shift to the neighboring category. If the majority of the cells was located in the same category at week 1 and 7, the clone was given the score 0. Less than 10% of clones containing the insulated vector were given the score −2. In contrast, near half of the SBT/RGIP clones moved from intermediate expression to the silent category (score −2). Seventy percent of the SBT/cHS4.RGIP.cHS4 clones had maintained or even increased expression (scores 0 and +1) after 7 weeks, whereas 32% of the clones carrying the uninsulated transposon maintained their initial level of eGFP expression (Figure 6b). In summary, our data demonstrate that insulators flanking the transgene expression cassette within DNA transposon-derived vectors protect against postintegrative silencing of transgene cassettes that are otherwise prone for transcriptional repression.

Figure 6.

Figure 6

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Silencing and insulation of SB transposon-based vectors in F9 cells. (a) Expression status of all clones determined at week 1 and 7 after transfection. Cells were transfected with pSBT/RGIP, or its derivatives, and selected for puromycin resistance (see Figure 5). Individual clones were expanded and then passaged for 7 weeks. Their eGFP expression status was determined at week 1 and 7. The number of clones with a certain expression status (N, negative; L, low expression; I, intermediate expression; H, high expression) is indicated by light gray and dark gray columns for measurements at week 1 and 7, respectively. The transposon vectors used are indicated below the x-axis (see legend to Figure 5 for explanation of the three constructs). (b) Stability of expression from insulated SB vectors. For each of the examined SB transposon vectors (indicated below the x-axis), the drift of expression in transposon-positive F9 clones is indicated. On the basis of the expression profiles determined at week 1 and 7, each clone was given a score according to its progression from one expression category to another. Clones with the score −2 (white columns) had moved two categories toward silencing (from ‘I' to ‘N' or ‘H' to ‘L'), whereas the scores -1 (light gray columns) and +1 (black columns) were given to clones in which reduction or increase of expression corresponded to a shift to a neighboring category. If the majority of the cells was located in the same category at week 1 and 7, the clone was given the score 0 (dark gray columns).

Discussion

By use of a nonselective method based upon PCR-based screening for stable transfectants, we demonstrate here that RSV-driven transgenes, genomically inserted in the context of SB DNA transposon–based vectors, are transcriptionally silenced in F9 teratocarcinoma cells. By screening of 243 F9 clones, each sorted on the basis of transient reporter gene expression, we found that transposition had occurred in 23% of the clones. In standard selection-based transposition assays, we could estimate that transposition occurred in at most 0.5% of the transfected cells. These data indicate that the true integration potential of SB is underestimated by approaches based on selection. Although we certified that only cells with low-to-intermediate transient eYFP expression were flow-sorted, we cannot exclude that this approach had selected for cells with a higher-than-average content of plasmid DNA. Nevertheless, a previous study in HeLa also described the discrepancy between detection of insertions by PCR and colony assays based on transgene expression,21 supporting the notion that a substantial portion of the transposon insertions both in HeLa and F9 cells does not result in levels of gene expression that can facilitate drug resistance. It is tempting, therefore, to suggest that most or some of these null insertions are transcriptionally silenced after integration. Indeed in our study, all clones containing a transcriptionally inactive RSV-eYFP transposon could be reignited by treatment of modulators of DNA methylation and histone acetylation, indicating that these particular insertions had in fact been epigenetically modified and silenced. Moreover, by following eYFP-expressing clones over time we found that a majority of these clones moved toward silencing for the duration of the experiment.

Considering the wealth of data describing silencing of retroviral vectors in F9 early embryonal cells and embryonic stem cells, we were interested in investigating the fate of transgenes delivered by transposons as an alternative gene insertion tool in F9 cells. Our data suggest that SB transposon–derived vectors do not intrinsically contain cis-elements that protect their content from pathways of transcriptional silencing in F9 cells. This finding seems to apply to transgenes driven by the viral RSV promoter and the widely active PGK promoter, both utilized in this article. Immediate transcriptional repression of retroviral vectors in F9 cells is triggered by a DNA element known as the repressor-binding site (RPS).24 The RPS sequence overlaps with the PBS sequence and is specifically recognized by a high-molecular weight complex that contains TRIM28.27 Recruitment of TRIM28 is correlated with silencing by a mechanism involving the recruitment of HP1γ and a specific interaction between TRIM28 and HP1γ.26 We searched for RPS-like sequences in our SB-based vectors but did not find sequences with homology to RPS. Although we cannot at this point exclude that mechanisms involving TRIM28 could come into play in repression of SB, activation of eYFP expression by treatment of silenced clones with 5-aza and/or the deacetylase inhibitor TSA seems to favor the notion that silencing is correlated with DNA methylation and histone deacetylation. Previous findings by Niwa and co-workers have indeed shown that the transcriptional stability of retroviral inserts is affected by DNA methylation.29 Moreover, it has been suggested that the high CpG content of GFP and related genes such as eYFP makes these genes susceptible to DNA methylation.48

Here, we set out to utilize the efficacious silencing of transgenes within SB-based vectors inserted in F9 cells as a model system for creating protected SB-derived gene carriers. Although previous findings have documented that insulators do not protect retroviral vectors from RPS-based repression of retroviral vectors in F9 cells,45 we aimed at characterizing the effects of such elements in a transposon context. In transposition assays based on selection, insertion of vectors with a transgene cassette flanked by 1.2-kb cHS4 insulators reproducibly led to higher levels of stable transfection. These data support the notion that the insulators increase the likelihood of SB-based expression at random chromosomal integration loci32 and have a profound effect on both RSV- and PGK-driven transgene expression. Although the presence of insulators could in theory increase the level of transfection and/or transposition from the donor plasmid, transposon excision assays did not favor this notion. Thus, it is most likely that the cHS4 sequences in the transposon insertion assay serve as barriers against position-effect variegation resulting on average in higher levels of expression of the puromycin resistance gene located within the transposed elements. In support of this hypothesis, we found that clones with initial expression of eYFP from the insulated SB vector were protected against transcriptional repression when the clones underwent prolonged passaging in culture. Hence, >60% of the clones carrying the cHS4-insulated transposon were stable over time, whereas >70% of the clones with the uninsulated variants were silenced or moved toward silencing. Despite these promising findings, our data also show that the insulators do not render absolute protection, as 12% of the SBT/cHS4.RGIP.cHS4 clones were indeed silenced. This conclusion is in concordance with results obtained with cHS4-insulated retroviral vectors in mouse progenitor cells.32,33,34 Transposition assays with vectors carrying shorter versions of the cHS4 insulator indicated that only the full-length 1.2-kb cHS4 insulator in our hands served as an efficient barrier element in the context of an SB vector. However, the extended 400-bp cHS4 core sequence has previously been shown to provide full barrier activity in hematopoietic progenitor cells.47 Although we cannot at this point explain these discrepancies, it remains a formal possibility that SB vectors containing the 400-bp extended core sequence did not transpose properly and therefore resulted in fewer puromycin-resistant colonies.

We show that cHS4 insulation of SB-based vectors provides a vector type that is more likely to express after insertion. Such vectors may have multiple applications in both transgenesis studies and efforts to facilitate therapeutic SB insertion in vivo. Our data also suggest that the transposon insertion itself is not the main trigger of repression, as insulators have a robust positive effect also on insertions that have not been catalyzed by the SB transposase. As would be expected, we found that silencing was particularly obvious in F9 clones, which were found by Southern blot analysis to contain a single insertion. To our surprise and continued scrutiny, we found that also clones containing multiple insertions (one clone with >6 insertions) were silenced. Perhaps this finding reveals a cumulative inhibitory effect of several insertions within a single cell, indicating that SB could possess self-regulatory mechanisms that affect silencing. Interestingly, we and others have recently shown that the terminal sequences of SB including the inverted repeats possess gene-regulatory cis-elements which direct transcription toward the center of the transposon.19,20 It may be hypothesized that double-stranded RNA, at levels above a threshold that is higher than that from a single or a few transposons, may in fact trigger a self-regulatory response that leads to silencing in F9 cells. Indeed, double-stranded RNA has been found to induce strong sequence-specific inhibition of gene expression in F9 cells by a mechanism that involves RNA processing by Dicer.30

For each application of SB-based gene transfer varying demands to promoter, transgene(s), type of cell or tissue, and transposition efficiency may influence the genetic vector design. It remains an attractive possibility to utilize CpG-free promoters and transgenes to avoid potential effects of DNA methylation. However, in gene therapy applications in particular, this may not always be possible, and insulators offer a well-documented approach for protecting the transgene cassette. Recent results suggest that the enhancer-blocking function of cHS4 insulators reduce the potential influence of the transgene cassettes on genes flanking the inserted SB vector.20 On the basis of combined beneficial effects of cHS4 insulators providing both increased safety and protection of the transgene cassette, we propose that cHS4-insulated vectors are considered as an option in future applications of the SB system in both transgenic animal development and gene therapy.

Materials and Methods

Plasmid construction. Because of the appearance of new DNA transposon– based vector systems, plasmids carrying SB transposon vectors are throughout this article designated pSBT/ (as opposed to the conventional pT/ designation). The plasmids pSBT/RSV-eYFP.CMV-SB (Figure 2), pCMV-SB, and pCMV-mSB have been described previously.1,6,21 The pSBT/PGK-puro plasmid (plasmid size: 5.3 kb; transposon length: 2.6 kb) (Figure 3a) was described in ref. 10 as pT/puro. pSBT/scs.PGK-puro.scs (plasmid size: 6.7 kb; transposon length: 4.0 kb) was constructed by replacing the PGK-puro cassette from the pSBT/PGK-puro plasmid (NotI/SpeI) with the 680-bp PCR-amplified scs insulator containing a 3′-prime restriction site linker for further cloning. The second scs insulator was inserted (NotI/NheI) into the restriction site linker. The PGK-puro cassette was PCR-amplified with primers allowing insertion by PacI/NheI digestion the linker between the two scs insulator elements. pSBT/cHS4.PGK-puro.cHS4 (plasmid size: 7.7 kb; transposon length: 5.0 kb) was generated by inserting two PCR-amplified 1.2-kb cHS4 insulators in the same orientation side-by-side (as described earlier for scs to generate pSBT/scs.PGK-puro.scs) and inserting the PCR-amplified PGK-puro cassette between the two insulators. pSBT/β-gal.PGK-puro.β-gal (plasmid size: 7.7 kb; transposon length: 5.0 kb) was created by inserting a PCR-amplified 2,400-bp fragment derived from an internal segment of the β-gal gene into NotI/SpeI- digested pSBT/PGK-puro. The PGK-puro cassette was PCR-amplified with primers containing MluI restriction sites and inserted into an internal restriction site (Bss HII) located near the middle of the β-gal segment. As a result, the vector contained β-gal fragments on both sides of the PGK-puro cassette with resemblance to the composition of the SBT/cHS4.PGK-puro.cHS4 transposon. pSBT/RGIP (plasmid size: 6.2 kb; transposon length: 3.5 kb) was constructed by PCR-amplifying the GFP-IRES-puro cassette from the pSBT/RSV-FGIP plasmid, which has previously been described in ref. 49. The RSV promoter and startcodon was linked to the cassette by overlap PCR. The resulting RGIP (RSV-GFP-IRES-puro) cassette was inserted into NotI/SpeI-digested pSBT/PGK-puro. pSBT/L-cHS4.RGIP plasmid (plasmid size: 7.4 kb; transposon length: 4.7 kb) was generated by insertion of the 1.2-kb cHS4 element into the NotI site of pSBT/RGIP. pSBT/cHS4.RGIP.cHS4 plasmid (plasmid size: 8.6 kb; transposon length: 5.9 kb) was created by inserting an RSV-eYFP cassette (PacI/NheI digestion) between the two insulators followed by a replacement of the eYFP gene with a PCR-amplified GFP.IRES.puro cassette (NcoI/NheI digestion). Primer sequences are available on request.

Cell culture and transposition assays. The F9 embryonal teratocarcinoma cells were initially derived from a spontaneous testicular tumor in mouse strain 129 originating from an engrafted 6-day-old embryo.50 F9 cells were maintained under normal tissue culture conditions (37 °C, 5% CO2) grown in Dulbecco's modified Eagle's medium (Lonza, Basel, Switzerland) containing 10% fetal calf serum (Lonza, Basel, Switzerland), 0.26 mg/ml glutamine, 54 ng/ml penicillin, and 36 µg/ml streptomycin. The F9 cells were grown on gelatine-coated material. The cells were seeded in 1-cm2 wells in the concentration 1 × 105 cells/well. Day one after the seeding, the cells were transfected in the ratio: 6 µl FuGENE 6 (Roche, Basel, Switzerland), 1.5 µg transposase plasmid, and 1.5 µg transposon plasmid. Transfections were performed according to manufacturer's directions. The selection medium containing 1 µg/ml puromycin (Sigma Aldrich, St Louis, MO) was added day 2 after transfection. The colonies were stained when visible with methylene blue in methanol. For generation of cell lines by selection F9 cells were seeded in 1-cm2 wells in the concentration 1 × 105 cells/well. Day one after the seeding, the cells were transfected in the ratio: 3 µl FuGENE 6, 1.0 µg transposase plasmid and 1.0 µg transposon plasmid and 2 µl FuGENE 6, 0.5 µg transposase plasmid and 0.5 µg transposon plasmid to lower the number of integrations per cell. Transfections were performed according to the supplied protocol. The selection medium containing 1 µg/ml puromycin was added day 2 after transfection; single clones were isolated and the cells were kept under selection for 1 week. The selection medium was replaced with standard medium, the clones were analyzed by flow-cytometry at week 1, and passaged for 7 weeks. Statistical analyses and comparisons of transposon vectors were performed using unpaired t-tests.

Transposon excision assay. To measure transposon mobilization HeLa cells were seeded in 6-well dishes (1 × 105 cells/well) and transfected with 1 µg pSBT/PGK-puro, pSBT/scs.PGK-puro.scs, pSBT/cHS4-PGK-puro-cHS4, or pSBT/cHS4.core-PGK-puro-cHS4.core together with 1 µg pCMV-SB10 or pCMV-mSB. Low-molecular weight DNA was extracted 2 days after transfection using the QIAprep miniprep kit (CA 91355; Qiagen, Valencia, Spain) according to the manufacturer's guidelines, except for a 1-h incubation period at 55 °C (0.6% SDS and 0.08 mg/ml proteinase K together with buffer P1) after resuspension of the cell pellet. Fifty nanogram of the extracted DNA was used as template for a PCR with primers 5′-CCATTCGCCATTCAGGCTGCGCAAC and 5′-CAGTAAGAGAATT ATGCAGTGCTGCC. An aliquot of the PCR was used as template for a second PCR using primers 5′-GCGAAAGGGGGATGTGCTGCAAGG and 5′-TCTTTCCTGCGTTATCCCCTGATTC. An aliquot of the second PCR was used as template for a third PCR using primers 5′-CGATT AAGTTGGGTAACGCCAGGG and 5′- CAGCTGGCACGACAGG TTTCCCG. Thirty cycles (95 °C 30 s, 65 °C 30 s, and 72 °C 1 min) were used in all three PCRs. The PCR bands were excised from the gel and sequenced to confirm that the amplified sequences were plasmid repair products. As a DNA input control of the first PCR, a plasmid backbone–specific PCR was performed using primers 5′-CAAGAGCAACTCGGTCGC and 5′-TCGTTGTCAGAAGTAAGTTGGC.

Flow-cytometric analysis. For fluorescence-activated cell sorting based on transient expression of eYFP, F9 cells were transfected with 1 µg pSBT/RSV-eYFP.CMV-SB DNA and flow-sorted 24 hours after transfection. The cells were seeded in 96-well dishes with a density of <1 cell per well. Cell colonies were trypsinized and expanded. After expansion for ~1 week, the clones were analyzed by flow cytometry to establish the initial expression level of eYFP expression. A FACSVAntageSE (BD, Franklin Lakes, NJ) Cell sorter was used for the sorting of cells based on their scatter properties and eYFP expression. A 150-mW laser (Coherent, Santa Clara, CA) with a wavelength of 488 nm was used for excitation. GFP or YFP signals were detected using a standard optical filter set (560 SP DM; 530/30 BP). All measurements were referenced to a negative control and measured under the same conditions. Cell lines were analyzed by flow cytometry and plotted in graphs containing predivided categories N (no expression), L (low expression), I (intermediate expression), and H (high expression). The ‘N' region contains cells in which the SB-carried transgene does not express or has been silenced, whereas the remaining categories contain cells that are positive for YFP or GFP expression.

Southern blot analysis. Genomic DNA was prepared from cell pellets following phenol–chloroform extraction and ethanol precipitation. 10 µg genomic DNA was EcoRI-digested overnight (EcoRI is a transposon single cutter and cleaves the bacterial backbone, which gives a distinct band in case of episomal plasmid DNA). The digested DNA was electrophoresed in a 0.8% agarose gel and transferred to a Hybond membrane (GE Healthcare, Buckinghamshire, UK) overnight using 20× SSC. The membrane was hybridized using an eYFP-specific [α-32P] dCTP-labelled probe overnight.

TSA and 5-aza treatment. For tests of eYFP reignition of silenced eYFP-negative clones, the clones were grown in the presence of TSA (Sigma Aldrich, St Louis, MO), in the presence of 5-aza, or in the presence of both drugs. The F9 clones were treated with 8-µmol/l 5-aza 36 hours before analysis by flow cytometry and/or 1,200 nmol/l TSA 24 hours before analysis.

Supplementary MaterialFigure S1. Initial eYFP expression profiles of all transposon-positive F9 clones as determined by flow cytometry.Figure S2. eGFP expression profiles over 7 weeks in F9 clones harboring uninsulated and insulated SB-derived vectors.

Supplementary Material

Figure S1.

Initial eYFP expression profiles of all transposon-positive F9 clones as determined by flow cytometry.

Click here for supplemental data (15.9MB, tiff)
Figure S2.

eGFP expression profiles over 7 weeks in F9 clones harboring uninsulated and insulated SB-derived vectors.

Click here for supplemental data (46.7MB, tiff)

Acknowledgments

We thank Hanne Jakobsen for technical assistance. We kindly acknowledge Gary Felsenfeld for providing the cHS4 insulator and Haini N. Cai for the scs element. This work was made possible through the generous support by the Danish Medical Research Council, the Danish Research Council for Technology and Production, the Novo Nordisk Foundation, the Carlsberg Foundation, the Danish Cancer Society, The Karen Elise Jensen Foundation, The Danish Heart Foundation (08-4-R64-A2017-B941-22456), Aage Bangs Foundation, the Arne Hansen Fund, and the EU (EU-FP6-STREP, contract number 018961). B.M. was funded by a grant from the Danish Cancer Society. N.S. has been supported by a grant from the Novo Scholarship Programme in Biotechnology and Pharmaceutical Sciences.

REFERENCES

  1. Ivics Z, Hackett PB, Plasterk RH., and , Izsvak Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell. 1997;91:501–510. doi: 10.1016/s0092-8674(00)80436-5. [DOI] [PubMed] [Google Scholar]
  2. Geurts AM, Wilber A, Carlson CM, Lobitz PD, Clark KJ, Hackett PB, et al. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol. 2006;6:30. doi: 10.1186/1472-6750-6-30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Collier LS, Carlson CM, Ravimohan S, Dupuy AJ., and , Largaespada DA. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature. 2005;436:272–276. doi: 10.1038/nature03681. [DOI] [PubMed] [Google Scholar]
  4. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG., and , Jenkins NA. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature. 2005;436:221–226. doi: 10.1038/nature03691. [DOI] [PubMed] [Google Scholar]
  5. Keng VW, et al. Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods. 2005;2:763–769. doi: 10.1038/nmeth795. [DOI] [PubMed] [Google Scholar]
  6. Yant SR, Meuse L, Chiu W, Ivics Z, Izsvak Z., and , Kay MA. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet. 2000;25:35–41. doi: 10.1038/75568. [DOI] [PubMed] [Google Scholar]
  7. Ehrhardt A, Xu H, Huang Z, Engler JA., and , Kay MA. A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase phiC31 and the Sleeping Beauty transposase. Mol Ther. 2005;11:695–706. doi: 10.1016/j.ymthe.2005.01.010. [DOI] [PubMed] [Google Scholar]
  8. Mikkelsen JG, Yant SR, Meuse L, Huang Z, Xu H., and , Kay MA. Helper-Independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo. Mol Ther. 2003;8:654–665. doi: 10.1016/s1525-0016(03)00216-8. [DOI] [PubMed] [Google Scholar]
  9. Ohlfest JR, Frandsen JL, Fritz S, Lobitz PD, Perkinson SG, Clark KJ, et al. Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system. Blood. 2005;105:2691–2698. doi: 10.1182/blood-2004-09-3496. [DOI] [PubMed] [Google Scholar]
  10. Yant SR., and , Kay MA. Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells. Mol Cell Biol. 2003;23:8505–8518. doi: 10.1128/MCB.23.23.8505-8518.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Liu L, Mah C., and , Fletcher BS. Sustained FVIII expression and phenotypic correction of hemophilia A in neonatal mice using an endothelial-targeted sleeping beauty transposon. Mol Ther. 2006;13:1006–1015. doi: 10.1016/j.ymthe.2005.11.021. [DOI] [PubMed] [Google Scholar]
  12. Liu L, Sanz S, Heggestad AD, Antharam V, Notterpek L., and , Fletcher BS. Endothelial targeting of the Sleeping Beauty transposon within lung. Mol Ther. 2004;10:97–105. doi: 10.1016/j.ymthe.2004.04.006. [DOI] [PubMed] [Google Scholar]
  13. Ohlfest JR, et al. Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma. Mol Ther. 2005;12:778–788. doi: 10.1016/j.ymthe.2005.07.689. [DOI] [PubMed] [Google Scholar]
  14. Ohlfest JR, Lobitz PD, Perkinson SG., and , Largaespada DA. Integration and long-term expression in xenografted human glioblastoma cells using a plasmid-based transposon system. Mol Ther. 2004;10:260–268. doi: 10.1016/j.ymthe.2004.05.005. [DOI] [PubMed] [Google Scholar]
  15. Lohe AR., and , Hartl DL. Autoregulation of mariner transposase activity by overproduction and dominant-negative complementation. Mol Biol Evol. 1996;13:549–555. doi: 10.1093/oxfordjournals.molbev.a025615. [DOI] [PubMed] [Google Scholar]
  16. Geurts AM, et al. Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther. 2003;8:108–117. doi: 10.1016/s1525-0016(03)00099-6. [DOI] [PubMed] [Google Scholar]
  17. Sijen T., and , Plasterk RH. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature. 2003;426:310–314. doi: 10.1038/nature02107. [DOI] [PubMed] [Google Scholar]
  18. Slotkin RK., and , Martienssen R. Transposable elements and the epigenetic regulation of the genome. Nature Rev. 2007;8:272–285. doi: 10.1038/nrg2072. [DOI] [PubMed] [Google Scholar]
  19. Moldt B, Yant SR, Andersen PR, Kay MA., and , Mikkelsen JG. Cis-acting gene regulatory activities in the terminal regions of sleeping beauty DNA transposon-based vectors. Human Gene Ther. 2007;18:1193–1204. doi: 10.1089/hum.2007.099. [DOI] [PubMed] [Google Scholar]
  20. Walisko O, Schorn A, Rolfs F, Devaraj A, Miskey C, Izsvák Z, et al. Transcriptional activities of the Sleeping Beauty transposon and shielding its genetic cargo with insulators. Mol Ther. 2008;16:359–369. doi: 10.1038/sj.mt.6300366. [DOI] [PubMed] [Google Scholar]
  21. Garrison BS, Yant SR, Mikkelsen JG., and , Kay MA. Postintegrative gene silencing within the Sleeping Beauty transposition system. Mol Cell Biol. 2007;27:8824–8833. doi: 10.1128/MCB.00498-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Strickland S., and , Mahdavi V. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell. 1978;15:393–403. doi: 10.1016/0092-8674(78)90008-9. [DOI] [PubMed] [Google Scholar]
  23. Strickland S, Smith KK., and , Marotti KR. Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell. 1980;21:347–355. doi: 10.1016/0092-8674(80)90471-7. [DOI] [PubMed] [Google Scholar]
  24. Barklis E, Mulligan RC., and , Jaenisch R. Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells. Cell. 1986;47:391–399. doi: 10.1016/0092-8674(86)90596-9. [DOI] [PubMed] [Google Scholar]
  25. Teich NM, Weiss RA, Martin GR., and , Lowy DR. Virus infection of murine teratocarcinoma stem cell lines. Cell. 1977;12:973–982. doi: 10.1016/0092-8674(77)90162-3. [DOI] [PubMed] [Google Scholar]
  26. Wolf D, Cammas F, Losson R., and , Goff SP. Primer binding site-dependent restriction of murine leukemia virus requires HP1 binding by TRIM28. J Virol. 2008;82:4675–4679. doi: 10.1128/JVI.02445-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wolf D., and , Goff SP. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell. 2007;131:46–57. doi: 10.1016/j.cell.2007.07.026. [DOI] [PubMed] [Google Scholar]
  28. Modin C, Lund AH, Schmitz A, Duch M., and , Pedersen FS. Alleviation of murine leukemia virus repression in embryonic carcinoma cells by genetically engineered primer binding sites and artificial tRNA primers. Virology. 2000;278:368–379. doi: 10.1006/viro.2000.0683. [DOI] [PubMed] [Google Scholar]
  29. Niwa O, Yokota Y, Ishida H., and , Sugahara T. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells. Cell. 1983;32:1105–1113. doi: 10.1016/0092-8674(83)90294-5. [DOI] [PubMed] [Google Scholar]
  30. Billy E, Brondani V, Zhang H, Muller U., and , Filipowicz W. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA. 2001;98:14428–14433. doi: 10.1073/pnas.261562698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Chung JH, Whiteley M., and , Felsenfeld G. A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell. 1993;74:505–514. doi: 10.1016/0092-8674(93)80052-g. [DOI] [PubMed] [Google Scholar]
  32. Rivella S, Callegari JA, May C, Tan CW., and , Sadelain M. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J Virol. 2000;74:4679–4687. doi: 10.1128/jvi.74.10.4679-4687.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Emery DW, Yannaki E, Tubb J., and , Stamatoyannopoulos G. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA. 2000;97:9150–9155. doi: 10.1073/pnas.160159597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yannaki E, Tubb J, Aker M, Stamatoyannopoulos G., and , Emery DW. Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors. Mol Ther. 2002;5:589–598. doi: 10.1006/mthe.2002.0582. [DOI] [PubMed] [Google Scholar]
  35. Li CL., and , Emery DW. The cHS4 chromatin insulator reduces gammaretroviral vector silencing by epigenetic modifications of integrated provirus. Gene Ther. 2008;15:49–53. doi: 10.1038/sj.gt.3303009. [DOI] [PubMed] [Google Scholar]
  36. Litt MD, Simpson M, Gaszner M, Allis CD., and , Felsenfeld G. Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science. 2001;293:2453–2455. doi: 10.1126/science.1064413. [DOI] [PubMed] [Google Scholar]
  37. Litt MD, Simpson M, Recillas-Targa F, Prioleau MN., and , Felsenfeld G. Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci. EMBO J. 2001;20:2224–2235. doi: 10.1093/emboj/20.9.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mutskov VJ, Farrell CM, Wade PA, Wolffe AP., and , Felsenfeld G. The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation. Genes Dev. 2002;16:1540–1554. doi: 10.1101/gad.988502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pikaart MJ, Recillas-Targa F., and , Felsenfeld G. Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev. 1998;12:2852–2862. doi: 10.1101/gad.12.18.2852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Recillas-Targa F, et al. Position-effect protection and enhancer blocking by the chicken β-globin insulator are separable activities. Proc Natl Acad Sci USA. 2002;99:6883–6888. doi: 10.1073/pnas.102179399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van der Vlag J, den Blaauwen JL, Sewalt RG, van Driel R., and , Otte AP. Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J Biol Chem. 2000;275:697–704. doi: 10.1074/jbc.275.1.697. [DOI] [PubMed] [Google Scholar]
  42. Caldovic L, Agalliu D., and , Hackett PB. Position-independent expression of transgenes in zebrafish. Transgenic Res. 1999;8:321–334. doi: 10.1023/a:1008993022331. [DOI] [PubMed] [Google Scholar]
  43. Dunaway M, Hwang JY, Xiong M., and , Yuen HL. The activity of the scs and scs' insulator elements is not dependent on chromosomal context. Mol Cell Biol. 1997;17:182–189. doi: 10.1128/mcb.17.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhong XP., and , Krangel MS. An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus. Proc Natl Acad Sci USA. 1997;94:5219–5224. doi: 10.1073/pnas.94.10.5219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Modin C, Pedersen FS., and , Duch M. Lack of shielding of primer binding site silencer-mediated repression of an internal promoter in a retrovirus vector by the putative insulators scs, BEAD-1, and HS4. J Virol. 2000;74:11697–11707. doi: 10.1128/jvi.74.24.11697-11707.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Izsvak Z, Ivics Z., and , Plasterk RH. Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates. J Mol Biol. 2000;302:93–102. doi: 10.1006/jmbi.2000.4047. [DOI] [PubMed] [Google Scholar]
  47. Aker M, et al. Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther. 2007;18:333–343. doi: 10.1089/hum.2007.021. [DOI] [PubMed] [Google Scholar]
  48. Dalle B, et al. eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides. Mol Ther. 2005;11:591–599. doi: 10.1016/j.ymthe.2004.11.012. [DOI] [PubMed] [Google Scholar]
  49. Moldt B, Staunstrup NH, Jakobsen M, Yanez-Munoz RJ., and , Mikkelsen JG. Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase. BMC Biotechnol. 2008;8:60. doi: 10.1186/1472-6750-8-60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Artzt K, Dubois P, Bennett D, Condamine H, Babinet C., and , Jacob F. Surface antigens common to mouse cleavage embryos and primitive teratocarcinoma cells in culture. Proc Natl Acad Sci USA. 1973;70:2988–2992. doi: 10.1073/pnas.70.10.2988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Initial eYFP expression profiles of all transposon-positive F9 clones as determined by flow cytometry.

Click here for supplemental data (15.9MB, tiff)
Figure S2.

eGFP expression profiles over 7 weeks in F9 clones harboring uninsulated and insulated SB-derived vectors.

Click here for supplemental data (46.7MB, tiff)

Articles from Molecular Therapy: the Journal of the American Society of Gene Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

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